Fig. 3: Pharmacological ATR inhibition has on-target activity and leads to reduced BRCA1 activation and repressed homologous recombination.
From: Therapeutic targeting of ATR in alveolar rhabdomyosarcoma
a Volcano plot showing relative changes in phospho-peptide abundance in PAX3-FOXO1-expressing Rh30 cells after 2 h of incubation with AZD6738 (750 nM) measured using LC-MS/MS proteomics (red, known ATR targets; dotted line indicating a false discovery rate (FDR) of 0.001). b Volcano plot showing relative enrichment of molecular pathways in which differential phospho-peptide abundance was observed in cells treated with AZD6738 (750 nM) compared to DMSO-treated cells (dotted line indicating a false discovery rate of 0.05). c Cellular processes significantly enriched in differentially abundant phospho-peptides. Western immunoblotting of BRCA1 S1524 and total BRCA1 in six FP-RMS cells after 2 hours of treatment with AZD6738 (750 nM) (d) or BAY 1895344 (20 nM) (e). f Quantification of changes in BRCA1 S1524 phosphorylation (P = 0.016; 0.016 for d and e, respectively; statistical analysis is sign test). g Relative HR activity in Rh4 and Rh30 cells after incubation with AZD6738 (750 nM), measured as GFP reconstitution based on repair of an SceI-mediated DNA lesion via homologous recombination. (n = 3 biologically independent experiments; P = 0.003; 2.61 × 10−6 for Rh4 and Rh30, respectively). h Excess over Bliss analysis of combined treatment with olaparib and AZD6738 in Rh4 cells (n = 3). i Bliss synergy scores for six FP-RMS cell lines treated with AZD6738 and olaparib. All statistical analyses correspond to two-sided student’s t-test except for 3f; data presented as mean value ± error bars representing standard deviation.
